Restoration of microRNA‐30b expression alleviates vascular calcification through the mTOR signaling pathway and autophagy

Abstract

Pathological calcification represents an event that consequently leads to a distinct elevation in the morbidity and mortality of patients with chronic kidney disease (CKD) in addition to strengthening its correlation with hyperphosphatemia. Epigenomic regulation by specific microRNAs (miRNAs) is reported to be involved in ectopic calcification. However, the finer molecular mechanisms governing this event remain unclear. Hence, this study aimed to identify the potential miRNAs involved in vascular calcification (VC) development and progression. Initially, mitochondrial membrane potential (MMP), autophagy‐specific markers (LC3II/LC3I and Beclin1) and phenotype‐specific markers of osteoblasts (runt‐related transcription factor 2 and Msx2) were measured to evaluate autophagy and VC in β‐glycerophosphate‐induced vascular smooth muscle cells (VSMCs) with either miR‐30b restoration or miR‐30b knockdown performed in vitro. The VC in vivo was represented by calcified nodule formation in the aorta of the rats undergoing 5/6 nephrectomy followed by a 1.2% phosphorus diet using Alizarin Red staining. SOX9 was verified as the target of miR‐30b according to luciferase activity determination. Restoration of miR‐30b was revealed to markedly diminish the expression of SOX9 while acting to inhibit activation of the mTOR signaling pathway. Knockdown of miR‐30b reduced MMP and autophagy, elevated VC, and suppressed the presence of rapamycin (an inhibitor of the mTOR signaling pathway). In addition, upregulated expression of miR‐30b attenuated VC in vivo. Taken together, the key findings of this study identified the inhibitory role of miR‐30b in VC, presenting an enhanced understanding of miRNA as a therapeutic target to curtail progressive VC in hyperphosphatemia of CKD.

RETRACTED: OGT-Mediated KEAP1 Glycosylation Accelerates NRF2 Degradation Leading to High Phosphate-Induced Vascular Calcification in Chronic Kidney Disease

“…Meanwhile, 62 rats successfully underwent VC development, and success rate was calculated to be 87.10% (54/62). Based on the standard two-step surgical ablation procedure, CKD was induced by 5/6 nephrectomy (5/6 Nx; Xu et al, 2019). The left kidney was simply removed by two-thirds.…”

“…Upon reaching 80-90% confluence, cells were detached in 0.25% trypsin (25200056, Gibco), followed by passaging. VC cell models were induced with 10 mmol/L β-glycerol phosphate (50020, Sigma-Aldrich, St Louis, MO, United States) for 9 days, whereas the control group was treated with equal volumes of phosphate buffer saline (PBS; Xu et al, 2019). β-glycerol phosphate-induced VSMCs were transduced with shRNAs targeting OGT, NRF2, or KEAP1, overexpression plasmids of OGT (oe-OGT), alone or in combination, as well as their controls.…”

miR-27a-3p Targets ATF3 to Reduce Calcium Deposition in Vascular Smooth Muscle Cells

“…For example, the miR-30 family has been reported to inhibit vascular calcification. 19 , 20 , 21 , 22 , 23 miR-125b 24 and miR-135a 24 are also anti-calcification miRNAs. In contrast, miR-32 , 24 miR-324-3p , 25 and miR-486 26 are examples of pro-calcification miRNAs.…”

The Epigenetic Landscape of Vascular Calcification: An Integrative Perspective

“…Among them, 15 (40.5%) have been confirmed or are suspected to promote or aggravate VC in different scenarios, while 22 (59.5%) protect against VC formation or progression. Direct gene targets have been identified for 14 miRNAs with negative VC regulatory capacity, including miR-29a/29b [14], miR-29b-3p [34], miR-30b [16,47], miR-30c [16], miR-30e [25], miR-34b/34c [29,64], miR-125b [13,22] miR-133a [19], miR-135a [30], miR-182 [42], miR-204 [15,41,50], and miR-205 [21], while few direct targets have been identified for miRNAs with VC enhancement ability (miR-34a [39], miR-128-3p [48], and miR-135a-3p [17]). In the following sections, we describe the clinical features of each VC-regulating miRNA based on their propensity for positive or negative vascular influences.…”

“…Louvet et al also reported that miR-30b was down-regulated during in vitro human VSMC calcification [28]. Other researchers used mouse or rat VSMC and tissues for analysis; they showed similarly that miR-30b and miR-30e over-expression alleviated VC, but the direct targets in murine VSMCs (insulin-like growth factor 2 [IGF2] and SOX9) seemed to differ from those (RUNX2 and Smad1) of human ones, and the mechanisms involved varied as well (mTOR pathway in murine cells) [25,47].…”